Migraine is an immense healthcare burden with limited therapeutic options. Environmental enrichment (EE) reduces migraine. The long-term goal of this project is to determine how EE mitigates migraine, modeled using spreading depression (SD), in order to identify novel targets for development of therapies. SD, the likely cause of migraine with aura (MwA), transiently disrupts myelin. Microglia can polarize to distinct phenotypes that range from classically (M1) to alternatively (M2a) activated states. Microglia generate the oxidative stress that occurs with SD and lowers SD threshold (SDT), suggesting that a pro-inflammatory M1 phenotype enhances SD by damaging myelin and increasing aberrant cross-talk between nerve fibers. In contrast, EE promotes an anti-inflammatory M2a phenotype and raises SDT, while increasing myelination, and oxidative tolerance (OT). EE increases CNS interleukin-11 (IL-11), and IL-11 alone can recreate these effects. The rationale for this project is based on evidence indicating that microglial M2a polarization increases SDT via secreted factors. Preliminary data show that while exosomes released from M1 microglia reduce SDT, those from M2a microglia increase SDT, perhaps by increasing myelination/OT, which could prevent the aberrant excitability that provokes SD. This study will further explore the effects of EE-induced M2a polarization on increasing SDT through production of exosomes. The overall goal is to define exosomal miRNAs from M2a- polarized microglia that most potently increase SDT plus myelination/OT and test their effectiveness in vivo after nasal administration. The central hypothesis is that M2a-polarized microglia release exosomes containing miRNA that mimic the effects of EE on SDT, myelin levels and OT. The aims are: Aim 1: Establish the impact of M2a versus M1 microglia-derived exosomes on SDT, myelination/OT. SD is used to model MwA in rat brain slice cultures and rats. Exosomes from polarized primary microglia are applied to slice cultures for measurement of SDT, myelination/OT. Aim 2: Identify miRNA species differentially expressed in M2a versus M1 microglia-derived exosomes and validate their role in influencing myelination/OT. miRNAs are identified using arrays and bioinformatics. Targets are verified using qPCR and Western blots. Aim 3: Determine the extent to which unique miRNAs identified in Aim 2 impact SDT, myelination/OT. Exosomes from M2a microglia and dendritic cells (DCs) will be electroporated with specific miRNA inhibitors and mimics respectively, then tested for impact on slice culture SDT, myelination/OT. DC exosomes will be used, as they enter brain nasally to produce functional effects. Aim 4: Establish the impact of M2a exosomes in vivo. M2a exosomes will be nasally delivered to brain for measurement of SDT, myelination/OT, and target mRNA/proteins. Trafficking will be determined via in vivo and confocal imaging of tagged exosomes. This work is innovative because it explores M2a microglial exosomes/miRNAs as a means to increase SDT via myelination/OT. It is significant, because it will identify therapeutic miRNAs to be loaded into exosomes for nasal delivery against SD/MwA.